In this paper, we evaluated culture conditions to optimize the proliferation of human LPCs that were prospectively isolated by cell surface markers using FACS sorting.18,36
Although the identity of the antigen recognized by the 5E12 monoclonal antibody is not yet known, it has been shown to be a relevant marker for progenitor cells in at least the neural lineage.16,36
This antibody enabled the enrichment of LPCs from human fetal liver (). These LPCs cultured on feeder layers of FFS were capable of giving rise to colonies of bipotential phenotype including both albumin and CK19-expressing cell characteristic of hepatocyte and biliary cells, respectively.18,36
Among the markers of advanced differentiation arising in these colonies are the receptors for infection by hepatitis D virus, as demonstrated in .
When these highly purified LPCs were cultured alone, under standard 2D conditions, there was minimal in vitro
expansion of these cells. Since others have reported that human fetal liver cells or partially purified fetal liver progenitors10–12,37
are able to expand when cultured under standard 2D conditions or cocultured with stromal cells such as FFS,9
we postulated that the highly purified LPC population excludes some cells that may support progenitor cell proliferation in vitro
We further postulated that these supporting cells may be endothelial cells based on the findings of others that liver development and regeneration have been demonstrated to be closely linked to angiogenic endothelial cells in vivo
To test this hypothesis, we cocultured highly purified LPCs with nonparenchymal endothelial cells in 3D fibrin gel in order to allow LPCs to utilize natural in vivo
mechanisms to facilitate their expansion and function in vitro
. We report that, compared to 2D culture conditions and to cultures of LPCs alone in 3D, LPCs cocultured with EC under 3D conditions exhibited increased expansion and increased and sustained production of albumin and AFP.
Our data supported our hypothesis and demonstrated that coculturing LPCs with endothelial cells in 3D fibrin gel enhanced the LPC expansion along with the formation of vascular structures. The LPCs had the capacity for further growth and expansion in 3D coculture, as demonstrated by the BrdU labeling (). LPC proliferation and production of albumin and AFP both were enhanced by coculture with HUVECs in 3D fibrin gel ( and ). The spilt-3D fibrin gel and transwell culture of LPCs and HUVECs indicated that the LPC–HUVEC direct contact is essential for LPC function (). Less frequently, some clusters of LPCs in these 3D cocultures were observed to not be directly associated with HUVECs, when examined in situ
by fluorescent or confocal microscopy. These findings of EC-dependent growth, even without continued direct contact, may be explained by a recent report that direct contact between hepatocytes and supportive stromal cells–fibroblasts, followed by a sustained short-range soluble signal, is enough for maintenance of the hepatocellular phenotype in coculture.39
The ability of both cells to survive and expand to the late time points, as described in our results, implies that LPCs and HUVECs had a reciprocal effect on each other's growth (). The vascular structures described in the 3D culture model in this paper, which occurred in a period of 1 to 2 weeks, are consistent with in vitro
angiogenesis model systems described by others.40–42
This structure, however, began to regress at day 36 and was almost undetectable by fluorescent microscopy at day 48. Nahmias et al
described a coculture system containing mature hepatocytes and endothelial cells on matrigel, in which mature hepatocytes were recruited to endothelial vascular structures by endothelial cell–derived hepatocyte growth factor and then formed a sinusoid-like structure. This sinusoid-like structure retained hepatic function up to 2 months.8
Interestingly, the stability of the sinusoid-like structures was dependent on the presence of dermal fibroblasts in culture.8
It remains to be address whether LPCs in our coculture system were contaminated by fetal liver–derived fibroblast-like cells and whether, if contaminated, those fibroblast-like cells have a similar effect on vascular structures as dermal fibroblasts had.
We did not address in this paper whether these vascular structures demonstrated functional characteristics of blood vessels, and limited ourselves to correlating the differences in LPC function with the structural events noted to occur with these endothelial cells. We did, however, confirm that these resulting vascular structures arose from EGFP-expressing HUVECs. We do not believe that the findings were specific to HUVECs in that freshly isolated endothelial cells from human fetal liver also enhanced the secretion of albumin and AFP when cocultured with LPCs (data not shown). How cells, including endothelial cells, LPCs, and maybe other types of cells, extracellular matrix, and cytokines/growth factors in 3D fibrin gel enhanced LPC proliferation and how the initiation, development, and regression of vascular structures were affected by LPC expansion and manipulations to optimize in vitro expansion in this model are the subjects of ongoing studies.
We selected fibrin gel as a polymer because it is a natural extracellular matrix and hydrogel and has already been used in clinical applications. Fibrin gel is derived from fibrinogen and does not contain other extracellular matrix and growth factors as matrigel does. The 3D fibrin gel has been proven to be suitable for an in vitro
and it is biocompatible and biodegradable, desirable characteristics for tissue engineering purposes.43
During preparation of 3D fibrin gels containing LPCs and endothelial cells, polymerization just requires a very mild enzymatic reaction condition and thus has no adverse effects on the cells. The firmness of fibrin gel is adjustable through changing fibrinogen concentration, and cell density in 3D fibrin is also changeable. The fibrin gel–containing cocultures, however, underwent obvious degradation beginning at the fourth week and eventually became degraded into an uneven stack of cells and extracellular matrix. The secretion of matrix metalloproteinases and plasmin in a coculture was the likely cause for fibrin gel degradation. The degradation of fibrin may affect long-term maintenance of hepatocyte culture in vitro
but is an attractive characteristic of a cell delivery vehicle for implants in vivo
Though there were some clusters formed in the 2D cocultures, these clusters were primarily located on the edges of the culture wells, which differ from the distribution of LPC clusters in the 3D coculture system (which were located throughout). The fibrin gel was relatively thicker at the edges of the 2D fibrin cultures than in the central counterpart. We therefore postulate that the thicker edges of the 2D fibrin gel allow for both formation of vascular structure similar to the 3D gels and the secondary effects of HUVECs on LPC growth and function.
In summary, we have successfully tested the hypothesis that isolated bipotential liver progenitor cells from human fetal liver, when cultured under 3D angiogenic conditions, demonstrate the enhanced proliferation in culture. We propose that these culture conditions also favor the development of an optimal microenvironment for LPC expansion including LPC–HUVEC contact, growth factors, and extracellular matrix. Our novel description of the LPC expansion along with the formation of vascular structures of endothelial cells in this 3D model might provide a unique advantage for engineered liver devices and antivirus screening.